Staining procedure for the detection of microcracks: application to ewe bone

Microcracks are one of the determinants of the bone strength and their accumulation may contribute to increased fracture risk. They are detected after bulk staining with various dyes, including basic fuschin, calcein and xylenol orange. The duration of staining usually varies across types of bone and species. The ewe is a large animal with a bone remodeling similar to humans, used as an animal model in bone histomorphometry studies. The aim of the present study was to determine the optimal conditions for bulk staining with xylenol orange of ewe bone. Xylenol orange 5mM in 70% ethanol was applied to iliac crest and vertebral biopsies for 2 or 15 days or 1, 2 or 3 months. After embedding, sections of 40, 50 and 80 μm thick were cut with either a precision diamond wire saw or a microtome. The staining was not visible after 2 or 15 days and was heterogeneous after 1 or 2 months. The quality of 40 and 50 μm thick sections was not preserved compared with those of 80 μm. Microcracks were suitably observed on ewe bone after bulk staining with xylenol orange for 3 months, in 80 μm thick sections. We conclude that the staining procedures should differ when examining ewe or human bone. This may be due to differences in bone matrix composition.